Combination of catechin and quercetin for pharmaceutical or dietary use

ABSTRACT

The invention relates to a composition for pharmaceutical or dietary use that possesses antioxidant activity and characterized in that it contains as active principle a combination of catechin quercetin, which exert a synergistic action when combined in mutual molar ratios selected within a critical range, from 6:1 to 3:1 mol of catechin:quercetin.

[0001] It is known that moderate consumption of red wine is associatedwith a decreased incidence of cardiovascular events (More, Medicine1986:65:245-67; Graziano, N. Engl. J. Med. 1993:329:1829-34).Constituents of red wine such as flavonoids have been considered to beinvolved in the aforementioned beneficial effects on the cardiovascularsystem on account of their ability to inhibit platelet function. Indeed,experimental studies in vivo on animals demonstrated that both red wineand grape juice reduced platelet activation in canine coronary arteriesaffected by stenosis. A similar effect was observed with flavonoidsisolated from red wine, including quercetin, indicating that theseconstituents of red wine were involved in eliminating the reduction inflow caused by platelet aggregation (Slane, Clin. Res. 1994; 42; 169A(abstr.)). Several studies in vitro have demonstrated that flavonoidssuch as resveratrol, quercetin and catechin inhibit plateletaggregation; however, one potential limitation of these studies arisesfrom the fact that the concentration employed to obtain this inhibitionwas too high. Accordingly, some authors have called into question theantiplatelet activity exerted in vivo by these constituents of red wine(Janssen, Am. J. Clin. Nutr. 1998; 67; 255-62). It should be noted thatresearch into the effects of flavonoids on platelet function has untilnow focused on each component considered individually; there has neverbeen an investigation of whether the flavonoids can act in combinationto inhibit platelet activation. Following the consumption of red wine,more than one flavonoid is circulating in the human body, so such asynergy might be relevant, in that lower concentrations of flavonoidsthan those studied previously might modulate platelet activity.

[0002] Another question concerning the antiplatelet effect of theflavonoids is their mechanism of action. Although the results of themajority of studies are in agreement that the flavonoids interact withthe metabolism of arachidonic acid, thus inhibiting the production ofthromboxane A₂, the mechanism on which this action is based has neverbeen studied. The flavonoids are phenolic compounds whose antioxidanteffects are correlated with the deoxidation of radicals rather than withchelation of the metal. It has been suggested that inhibition both ofplatelet function and of metabolism of arachidonic acid depends on theantioxidant activity, but no study envisaged investigations to discoverwhether the flavonoids interact with platelet activation by contrastingthe effect of oxidizing species formed in situ. The present inventionwas therefore based on investigating whether the flavonoids, or some ofthem selectively, could act synergistically to inhibit plateletfunction, and to interfere with platelet function on the basis of anantioxidant effect.

[0003] As a result of this study, the present invention proposes acomposition for pharmaceutical or dietary use that possesses highantioxidant activity, characterized in that this active principlecomprises a combination of catechin and quercetin in the molar ratio inthe range between approx 6:1 and 3:1, respectively.

[0004] According to the invention, it has in fact been found,surprisingly, that these two specific flavonoids combined in the saidconcentration ratios are able to exert their antioxidant activitysynergistically.

[0005] For a better understanding of the characteristics and advantagesof the invention, the details of the study that led to it are nowdescribed.

SUBJECTS AND METHODS MATERIALS

[0006]³²Pi and [³H]oleic acid were from Amersham (Arlington Heights,Ill.). Fura 2/AM and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) werefrom Molecular Probes (Eugene, Oreg.) and Sepharose 2B was fromPharmacia (Uppsala, Sweden). The tetrapeptide Arg-Gly-Asp-Ser (RDGS) wasfrom Bachem Feinchemikalien AG (Budendorf, Switzerland). The type 1collagen was from Mascia Brunelli (Milan, Italy). The HPLC columns(Partisil 10 SAX) were from Whatman (Clifton, N.J.). The bovine serumalbumin, HEPES, acetylsalicylic acid, catechin, quercetin, fibrinogen,inorganic pyrophosphatase, digitonin, formaldehyde, indomethacin,phosphocreatine and creatine kinase were from Sigma Chemical Co. (St.Louis).

PLATELET PREPARATIONS

[0007] Drug-free human blood obtained from healthy volunteers wascoagulated with acid:citrate:dextrose. Platelet-rich plasma wascentrifuged at 800 x g for 20 min at room temperature and the pellet wassuspended in a volume equal to half the initial volume of autologousplasma, low in platelets. The platelet suspensions were incubated for 1h at 37° C. with 3 μmol of Fura 2/AM per L, 40 μmol DCFH-DA/L, 7.4 GBq(2 Ci) ³²Pi/L, or 3.7 MBq (1 mCi) [³H]oleic acid/L. The platelets werewashed by exclusion chromatography on Sepharose 2B using a Ca²⁺-freeTyrodes buffer (134 mmol NaCl/L, 2.9 mmol KCl/L, 0.34 mmol Na₂HPO₄/L and2 mmol MgCl₂/L) containing 0.2% of bovine serum albumin, 5 mmolglucose/L and 10 mmol HEPES/L, pH 7.35. The platelets that had beensubmitted to exclusion chromatography (PSEC) were adjusted to a finalconcentration of 2×10¹¹ cells/L. Since the addition of methanol to thesuspensions of PSEC at concentrations <0.5% did not cause any change inthe response of the PSEC to collagen, this ratio was used for obtainingfinal concentrations of quercetin that varied between 5 and 20 μmol/L.Catechin and quercetin were added to the suspensions of PSEC whilestirring continuously for 30 min at 37° C. and then removed bycentrifugation at 800 x g for 20 min at room temperature.

ANALYSIS OF PLATELET FLOW AND AGGREGATION BY CYTOMETRY

[0008] DCFH-DA was added to the PSEC (final concentration: 40 μmol/L);after 15 minutes of incubation with or without catechin or quercetin,the PSEC were activated with collagen. The reaction was stopped with 2mmol EGTA/L after 1 min. The samples were analysed in a Coulter XL-MCLflow cytometer (Hialeah, Fla.) equipped with an argon laser (emission480 nm) set up for measuring the logarithmic diffusion of direct light,which is a measure of the dimensions of the particle; logarithmicdiffusion of light at 90°, which is a measure of the granularity of thecell; and green fluorescence (DCF) 510-550 nm. The fluorescence signalgenerated by the probe was expressed as the stimulation index, i.e.intensity of mean channel fluorescence of the stimulatedplatelets/intensity of mean channel fluorescence of the unstimulatedplatelets. Platelet aggregation in vitro was evaluated according toBorn. The collagen was used at concentrations of 2-4 mg/L.

CONCENTRATIONS OF CYTOSOL PLATELET Ca²⁺

[0009] The concentrations of cytosol platelet Ca²⁺ were measured usingthe fluorescent indicator dye Fura 2, according to Grynkiewicz et al.;the changes in fluorescence were then monitored with a fluorimeter SFM25 (Kontron, Zurich, Switzerland) set at emission wavelength of 510 nmand excitation wavelength of 340 nm.

ACTIVATION OF PHOSPHOLIPASE C—PLATELET ADHESION TO COLLAGEN

[0010] The production (1,3,4-inositol triphosphate (IP₃), an indicatorof activation of phospholipase C, was analysed 30 s after plateletstimulation according to Pulcinelli et al. The collagen was used at aconcentration of 10 mg/L, which was the lowest concentration capable ofinducing a reproducible response. The platelet suspensions TABLE 2Case-control studies of alcohol consumption and coronary artery diseaseAlcohol Relative Reference Population Cases Controls Measure* Risk†Adjusted For 145 Boston Collaborative Drug Hospitalized, nonfatalHospitalized ≧6 drinks/day 0.6 (0.3-1.1) Age, sex, cigarette useSurveillance Program. M MI vs. and W, age 40-69. <6 drinks/day 1.0 66Two-Florida Counties Death from CAD Living Any alcohol 0.6 (0.5-0.8)Cigarette use, weight, religion Study. M. community vs. No alcohol 1.088 Kalser-Permanante, San Hospitalized, first MI Hospitalized 6drinks/day 0.4 (p < .01) Cigarette use, age, sex, weight, Francisco. Mand W. vs. BP, cholesterol 3-5 drinks/day 0.7 (p < .05) vs. <3drinks/day 1.0 123 Walnut Creek Contracep- Myocardial infarction Non-MI,same Non-drinker 3.1 (1.6-6.0) Cigarette use, hypertension, tive DrugStudy. W, age (acute) year of birth vs. hyper-cholesterolemia, obesity18-54. drinker 1.0 and gall-bladder disease 127 Glasgow Blood PressurePast history myocar- No history of MI Nil or occasional 3.4 (1.2-9.9)Age, weight, BP, serum Clinic. Hypertensive M, dial infarction vs.cholesterol age 50-59. frequent or heavy 1.0 134 W, age 30-49HospitalIzed, first MI Hospitalized Current drinkers 0.7 (0.5-1.0) Age,hospital, cigarette use, lipid vs. levels, hypertension, obesity, neverdrinkers 1.0 oral contraceptive use 135 Los Angeles retirement Fatal CADCommunity 2 drinks/day 0.4 (p < .01) No adjustment community. W, ≦ age80 vs. no alcohol 1.0 84 M, age 30-54 Hospitalized, nonfatalHospitalized 1-7 drinks/day 1.2 (0.8-1.8) Cigarette use first MI vs.never drinkers 1.0

FIG. 3

[0011] Mean value (±SEM) of the percentage changes (Δ) in theconcentrations of intraplatelet calcium at the reference line, afterstimulation with collagen alone, and after stimulation with 4 mg ofcollagen/L (A) or 8 mg of collagen/L (B) with catechin (Cat; 50 and 100μmol/L), quercetin (Q; 10 and 20 μmol/L), or Cat (25 μmol/L)+Q (5μmol/L). n=5 tests. *¹⁹⁰ Significantly different relative to collagenalone: *P<0.05, ^(#)P<0.01.

FIG. 4

[0012] Mean value (±SEM) of the percentage changes (Δ) in the formationof 1,3,4-inositol triphosphate (IP₃) in the platelets at the referenceline, after stimulation with collagen alone, and after stimulation with10 mg of collagen/L (A) or 20 mg of collagen/L (B) with catechin (Cat;50 and 100 μmol/L), quercetin (Q; 10 and 20 μmol/L), or Cat (25μmol/L)+Q (5 μmol/L). n=5 tests. *^(#)Significantly different relativeto collagen alone: *P<0.05, ^(#)P<0.01.

FIG. 5

[0013] Mean value (±SEM) of the percentage changes (A) in plateletadhesion to collagen at the reference line, after stimulation withcollagen alone, and after stimulation with 50 mg of collagen/L withcatechin (Cat; 50 and 100 μmol/L), quercetin (Q; 10 and 20 μmol/L), orCat (25 μmol/L)+Q (5 μmol/L). n=5 tests. ^(#)Significantly differentrelative to collagen alone: P<0.01.

RESULTS ANALYSIS BY FLOW CYTOMETRY

[0014] Flow cytometry makes use of the properties of DCFH-DA, whichdiffuses rapidly through the cellular membranes and is then trappedinside the cell through a reaction of deacetylation. In the presence ofhydrogen peroxide, this compound is oxidized to dichlorofluorescein(DCF), which is highly fluorescent. The effect of scalar concentrationsof quercetin and catechin on the production of hydrogen peroxide inducedby 10 and 20 mg of collagen/L is shown in FIG. 1. Compared withuntreated platelets, the platelets stimulated with collagen increasedthe production of hydrogen peroxide, which depended on the concentrationof collagen used. Catechin and quercetin inhibited the production ofhydrogen peroxide caused by the collagen on the part of the platelets.The combination of 5 μmol of quercetin/L and 25 μmol of catechin/L gavea significant reduction in formation of hydrogen peroxide caused by 10and 20 mg of collagen/L; neither of the two compounds alone at such lowquantities is reported to have had any inhibitory effect.

PLATELET AGGREGATION

[0015] The effect of catechin and quercetin on platelet aggregation wasmeasured using two different concentrations of collagen. Both catechinand quercetin inhibited platelet aggregation caused by collagen. Thedegree of inhibition depended on the concentration of collagen used.Thus, 100 μmol of catechin/L inhibited ≈75% of platelet aggregationinduced by 2 mg of collagen/L and inhibited ≈39% of platelet aggregationinduced by 4 mg of collagen/L. In platelets treated with 20 μmol ofquercetin/L, the degree of inhibition of platelet aggregation induced bycollagen (2 and 4 mg/L) was 50% and 43% respectively. The combination of25 μmol of catechin/L and 5 μmol of quercetin/L, which had no influenceon platelet aggregation when used on their own, produced significant(55%) inhibition of platelet aggregation induced by both theconcentrations of collagen (FIG. 2).

CHANGES IN INTRACELLULAR CALCIUM CONCENTRATION

[0016] Catechin and quercetin inhibited the mobilization of calcium,expressed as a percentage change in the concentration of intracellularcalcium. In the platelets stimulated with 4 mg of collagen/L, 100 μmolof catechin/L and 20 μmol of quercetin/L produced a significant decreasein calcium mobilization, of 71% and 65% respectively.

[0017] Incubation of the platelets with 25 μmol of catechin/L plus 5μmol of quercetin/L according to the invention produced a significantinhibition of calcium mobilization of 71%. A similar result was alsoobserved when calcium mobilization was induced by 8 mg of collagen/L(FIG. 3).

ACTIVATION OF PHOSPHOLIPASE C

[0018] Production of [³²P]IP₃ in platelets stimulated by collagen wasinhibited by catechin and quercetin: 10 mg of collagen/L, 100 μmol ofcatechin/L and 20 μmol of quercetin/L caused a significant decrease inIP₃ production, of 50% and 93% respectively. Incubation of the plateletswith 25 μmol of catechin/L plus 5 μmol of quercetin/L according to theinvention caused a significant inhibition of IP₃ production of 72%;similar effects were observed when the platelets were stimulated with 20mg of collagen/L (FIG. 4), but the degree of inhibition was lower,though still significant.

PLATELET ADHESION TO COLLAGEN

[0019] The activation of platelets by collagen is a multistage process.Thus, after being attached initially to the platelets via the pathwaysof the second messenger, collagen stimulates the release of thromboxaneand ADP, which are important platelet agonists that induce aggregation.To study the adhesion of the platelets to collagen (50 mg/L) without theinterference of aggregation and of the activation induced by all theknown agonists released by the platelet granules on stimulation bycollagen, the platelets were subjected to preincubation with aspirin, acyclooxygenase inhibitor, with the ADP removal system phosphocreatineand creatine kinase, and with the fibrinogen-fibronectin antagonist RDGS(13).

[0020] The adhesion of the platelets to 50 μmol of collagen/L in thepresence of catechin (50 and 100 μmol/L), quercetin (10 and 20 μmol/L),and catechin (25 μmol/L) plus quercetin (5 μmol/L) according to theinvention is presented in FIG. 5. Catechin or quercetin on their owninhibited platelet adhesion to collagen, which was suppressedsignificantly by 100 μmol catechin/L and 20 μmol quercetin/L. Incubationof the platelets with 25 μmol of catechin/L plus 5 μmol of quercetin/Lproduced significant inhibition of platelet adhesion of 85%.

[0021] The following general conclusions can be drawn from the studydescribed above.

[0022] As already mentioned, in the prior art the relationship betweenconsumption of red wine and inhibition of platelet function has beenobserved in various experimental studies. In fact, intragastricadministration of 4.0 mL of red wine/kg of body weight suppressedplatelet activation completely in a canine model of coronary stenosis.Although the concentrations of flavonoids in the peripheral blood havenot been measured after the administration of red wine, other studies onthe same experimental model showed that the flavonoids inhibitedplatelet activation, thus suggesting their possible involvement in theinhibition of platelet function. According to the study of the presentinvention, incubation of the platelets with 5 μmol of quercetin/L plus20 μmol of catechin/L, which had no effect on platelet functionindividually at these concentrations, is associated with significantinhibition of platelet activation. It should be pointed out thatalthough stimulation was carried out with high concentrations ofcollagen (8-20 mg/L), necessary for identifying the mobilization ofcalcium and the production of IP₃, the combination of quercetin andcatechin inhibits platelet function in every case.

[0023] The combination of catechin and quercetin causes even moreprofound effects on platelet adhesion, which is suppressed almostcompletely when the platelets are treated according to the invention. Inview of the biological importance of platelet adhesion to collagen inthe initiation and progression of the arteriosclerotic process, theinvention is expected to be useful in particular in the treatment orprevention of cardiovascular disorders (arteriosclerosis, thrombosis,infarction, etc.), for improving cerebral functionality, and fortreating mental deterioration in old age.

[0024] Other useful indications, based fundamentally on the antioxidantand free-radical-scavenging activity of the active principle, comprisethose for the treatment or prevention of cellulite, skin ageing andwrinkles, hair loss, for counteracting the action of UV radiation and ofenvironmental pollutants.

[0025] As an overall conclusion, the invention demonstrates that theflavonoids quercetin and catechin act synergistically according to theconcentrations indicated for inhibiting platelet adhesion to collagenand platelet aggregation caused by collagen, opposing the intracellularproduction of hydrogen peroxide.

[0026] Non-limiting examples of practical application of pharmaceuticalor dietary compositions according to the present invention are nowdescribed.

[0027] It should be explained that, still in a non-limiting manner,these examples relate to an active principle consisting of acatechin-quercetin combination in molar ratio of approx 5:1.

[0028] In particular, the said combination of the two flavonoids, called“complex” in the examples, is obtained according to the examples from anextract of parts, such as seeds and leaves, of Vitis vinifera containingapprox. 7.5 g of catechin and 1.5 g of quercetin per 100 g of extract.These compositions are preferably taken on a full stomach to optimizethe bioavailability of the active principle.

EXAMPLE 1 DIETARY PRODUCT FOR PREVENTING AND COMBATING CELLULITE SoftGelatin Capsules

[0029] Composition Each soft gelatin capsule (pearl) contains:Catechin + Quercetin complex 60 mg Ginkgo biloba dry extract with 24% of15 mg ginkgoflavonglucosides Centella asiatica, triterpene fraction 10mg Orthosiphon stamineus, dry extract 75 mg Fucus vesiculosus with 0.1 %of iodine 100 mg Linden sapwood 50 mg Chromium-containing yeast 12.5 mg(equal to chromium 0.015 mg) Vitamin E acetate 7.5 mg Soya oil 290 mgSoya lecithin 5 mg Mono- and diglycerides of fatty acids 30 mg Gelatin144 mg Glycerol 66 mg Iron oxide 0.3 mg Titanium dioxide 2.3 mgClorofilia rameica 0.5 mg

EXAMPLE 2 DIETARY PRODUCT FOR PREVENTING AND COMBATING CELLULITE Sachetsto be Dissolved in Water

[0030] Composition Each sachet contains: Catechin + Quercetin complex100 mg Ginkgo biloba dry extract with 24% of 15 mgginkgoflavonglucosides Centella asiatica, triterpene fraction 10 mgOrthosiphon stamineus, dry extract 100 mg Fucus vesiculosus at 0.1% ofiodine 100 mg Linden sapwood 100 mg Chromium-containing yeast 12.5 mg(equal to chromium 0.025 mg) Vitamin E acetate 7.5 mg Maltodextrin 2000mg Sodium citrate 360 mg Citric acid monohydrate 200 mg Tropical aroma120 mg Sour cherry aroma 60 mg Colloidal silica 70 mg Acesulfame K 8 mgAspartame 33 mg

EXAMPLE 3 PRODUCT FOR REINFORCING THE HAIR AND REDUCING HAIR LOSS SoftGelatin Capsules

[0031] Composition Each soft gelatin capsule (pearl) contains:Catechin + Quercetin complex 60 mg Methylsulphonylmethane 100 mg VitaminC 45 mg Vitamin E acetate 7.5 mg Zinc (as amino acid chelate) 3.75 mgCopper (as amino acid chelate) 0.625 mg Vitamin B6 1.0 mg Calciumpantothenate 4.5 mg Folic acid 0.15 mg Biotin 0.075 mg Spermidine 0.25mg Soya oil 290 mg Soya lecithin 5 mg Mono- and diglycerides of fattyacids 30 mg Gelatin 145 mg Glycerol 65 mg Titanium dioxide 2.8 mg Ironoxide 0.1 mg Clorofilla rameica 0.6 mg

EXAMPLE 4 COMPOSITION FOR PREVENTING CARDIOVASCULAR DISEASES SoftGelatin Capsules

[0032] Composition Each soft gelatin capsule (pearl) contains:Catechin + Quercetin complex 100 mg Ubidecarenone 10 mg Carnitine 100 mgEicosapentaenoic acid (EPA) 300 mg Docosahexaenoic acid (DHA) 200 mgLutein 2 mg 5-Methyltetrahydrofolic acid 010 mg Soya oil 250 mg Soyalecithin 10 mg Mono- and diglyceridcs of fatty acids 40 mg Gelatin 150mg Glycerol 70 mg Titanium dioxide 2.5 mg Iron oxide 0.2 mg Ladybird Red0.5 mg

EXAMPLE 5 DIETARY PRODUCT FOR PREVENTING SKIN AGEING AND WRINKLES SoftGelatin Capsules

[0033] Composition Each soft gelatin capsule (pearl) contains:Catechin + Quercetin complex 100 mg Lysine hydrochloride 125 mg VitaminC 45 mg Methylsulphonylmethane 100 mg Vitamin E acetate 7.5 mg Copper(as amino acid chelate) 0.625 mg Zinc (as amino acid chelate) 3.75 mgBiotin 0.075 mg Soya oil 290 mg Soya lecithin 5 mg Mono- anddiglycerides of fatty acids 30 mg Gelatin 145 mg Glycerol 67 mg Titaniumdioxide 2.5 mg Iron oxide 0.4 mg

EXAMPLE 6 DIETARY PRODUCT FOR IMPROVING CEREBRAL FUNCTIONALITY ANDPREVENTING MENTAL DETERIORATION IN OLD AGE Soft Gelatin Capsules

[0034] Composition Each soft gelatin capsule (pearl) contains:Catechin + Quercetin complex 100 mg Huperzine 0.050 mgPhosphatidylserine 50 mg Ginkgo biloba extract with 24% 15 mg ofginkgoflavonglucosides Vitamin B1 1.0 mg Vitamin B6 1.0 mg Vitamin B120.001 mg Vitamin C 90.0 mg Vitamin E acetate 7.5 mg Zinc (as amino acidchelate) 3.75 mg Copper (as amino acid chelate) 0.625 mg Soya oil 250 mgSoya lecithin 10 mg Mono- and digiycerides of fatty acids 40 mg Gelatin145 mg Glycerol 67 mg Titanium dioxide 1.5 mg Iron oxide 0.2 mg BluePatent V 0.5 mg

[0035] Other suitable pharmaceutical or dietary forms according to theinvention can be selected from a wide range, which includes everysuitable oral form such as tablets, granules, powders and others.

1. A composition for pharmaceutical or dietary use possessingantioxidant activity and characterized in that it contains as activeprinciple a combination of catechin and quercetin in molar ratio in therange between approx. 6:1 and 3:1, respectively.
 2. A compositionaccording to claim 1, characterized in that the said catechin andquercetin are in the ratio of approx. 5:1, respectively.
 3. Acomposition according to claim 1, characterized in that it is indicatedfor use as an agent for preventing platelet aggregation in the treatmentand prevention of cardiac and circulatory disorders.
 4. A compositionaccording to claim 1, characterized in that it is indicated forimproving cerebral functionality, in particular in the prevention andtreatment of mental deterioration in old age.
 5. A composition accordingto claim 1, characterized in that it is indicated for use in thetreatment and prevention of cellulite.
 6. A composition according toclaim 1, characterized in that it is indicated for use in the treatmentand prevention of skin ageing and wrinkles.
 7. A composition accordingto claim 1, characterized in that it is indicated for use in thetreatment and prevention of hair loss.
 8. A composition according toclaim 1, characterized in that it is indicated for use in the treatmentand prevention of skin damage caused by UV radiation.
 9. A compositionaccording to claim 1, characterized in that the said active principle isderived from an extract from parts, such as seeds and leaves, of Vitisvinifera.
 10. A composition according to claim 9, characterized in thatin the said extract there is approx 7.5 g of catechin and 1.5 g ofquercetin per 100 g of extract.
 11. Use of a combination of catechin andquercetin in the molar ratio in the range between 6:1 and 3:1,respectively, as active principle in the preparation of a compositionfor pharmaceutical or dietary use that possesses antioxidant activity.